Targeting the cell cycle for prognosis and therapy of breast cancer

Cyclin E is a G₁-cyclin that plays a key role in the G₁ to S transition of the cell cycle. Cyclin E is processed in tumor cells by an elastase-like protease into low-molecular-weight (LMW) isoforms that are biochemically hyperactive. The LMW isoforms of cyclin E are unique to cancer cells. In breast cancer, such alteration of cyclin E is a very strong predictor of poor patient outcome.

Alterations in the binding properties of these LMW isoforms to CDK2 and the CDK inhibitors (CKIs), p21 and p27, result in their functional hyperactivity. The LMW forms of cyclin E are several-fold more effective at binding to CDK2. Additionally, compared with the full-length cyclin E–CDK2 complexes, the LMW cyclin E–CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the CKIs to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity. When overexpressed in breast cancer cells, the LMW forms of cyclin E, but not the full-length form, result in their hyperactivity due to increased affinity for cdk2 and resistance to inhibition by the CDK inhibitors p21 and p27, result in resistance to the growth inhibiting effects of anti-estrogens, and result in chromosomal instability. Finally, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy.

To assess the oncogenic role of cyclin E-LMW as compared with full-length cyclin E, we examined the consequences of overexpressing these isoforms in the mammary glands of transgenic mice using the MMTV promoter. Four constructs were generated: MMTV-M46A coding for the full-length cyclin E (EL1), MMTV-EL1/EL4 coding for EL1 and the isoform translated at methionine 46 (EL4), and MMTV-T1 and MMTV-T2 coding for the isoforms generated by elastase cleavage at the first site (EL2 + EL3) and at the second site (EL5 and EL6), respectively. For each construct at least two transgenic lines were established. Transgene expression was demonstrated by RT-PCR, northern blotting and western blotting. Overexpression of cyclin E was seen in more than 90% of ductal and lobular cells of the mammary glands for each independent line. Mammary-specific LMW cyclin E overexpression induced extensive abnormalities at 2 months, including perturbed architecture, polyploidy, anysocytosis and apoptosis. Whole-mount preparations of mammary glands at different development stages showed that overexpression of EL1/EL4 and cyclin E-T1 induced growth delay, while at 6 months of age an increased proportion of cells in the S phase was found (25.6 ± 5.6% for EL1/EL4, 9.0 ± 2.7% for T1 compared with 3.9 ± 1.9% for non-transgenic animals). We observed a 34% (13/38) incidence of mammary adenocarcinomas in the EL1/EL4 transgenic lines with a mean latency of 18.3 months, and observed a 20% (5/25) incidence in the T1 transgenic lines with a mean latency of 17.1 months. The tumor incidence rate of the other transgenic lines, M46A and T2, are still unknown due to the young age of the mice (all under 7 months of age) and the long latency of cyclin E-mediated tumor generation. Thirty percent (4/13) of the EL1/EL4 and 40% (2/5) of the T1 tumor-bearing animals developed lung metastasis. The tumors induced by the EL1/EL4 and T1 transgenes were mainly solid adenocarcinomas with very little differential to glandular for EL1/EL4 and mostly glandular for T1. Since p53 alterations are common in human breast carcinomas, we bred a T1 line with p53^+/- mice. The T1 × p53^+/- cross generated tumors that are much more malignant than the T1 tumors; the incidence increased to 100%, with a much shorter latency of 11 months. Biochemical analysis of the tumors revealed that 64% (9/14) retained cyclin E expression and that, on average, the cyclin E-overexpressing tumors had threefold higher cyclin E kinase activity than the non-cyclin E-expressing tumors. Taken together, these data indicate that tumor progression in cyclin E transgenic mice follow sequential steps of dysplasia, mammary intraepithelial neoplasia and invasive/metastatic tumors.

Collectively, the biochemical and biological differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. The transgenic mouse model system can serve as a useful system in which to study the mechanisms responsible for LMW cyclin E-induced genetic instability and may help identify those factors that promote tumor progression and metastasis. The properties of the LMW forms of cyclin E suggest that they are not just surrogate markers of poor outcome, but that they are bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.